Muscarinic Receptor Stimulation Does Not Inhibit Voltage-dependent Ca²⁺ Channels in Rat Adrenal Medullary Chromaffin Cells

Abstract

Adrenal medullary chromaffin (AMC) and sympathetic ganglion cells are derived from the neural crest and show a similar developmental path. Thus, these two cell types have many common properties in membrane excitability and signaling. However, AMC cells function as endocrine cells while sympathetic ganglion cells are neurons. In rat sympathetic ganglion cells, muscarinic M₁ and M₄ receptors mediate excitation and inhibition via suppression of M-type K⁺ channels and suppression of voltage-dependent Ca²⁺ channels, respectively. On the other hand, M₁ receptor stimulation in rat AMC cells also produces excitation by suppressing TWIK-related acid sensitive K⁺ (TASK) channels. However, whether M₄ receptors are coupled with voltage-dependent Ca²⁺ channel suppression is unclear. We explore this issue electrophysiologically and biochemically. Electrical stimulation of nerve fibers in rat adrenal glands trans-synaptically increased the Ca²⁺ signal in AMC cells. This electrically evoked increased Ca²⁺ signal was not altered during muscarine-induced increase in Ca²⁺ signal, whereas it decreased significantly during a GABA-induced increase, due to a shunt effect of increased Cl⁻ conductance. The whole-cell current recordings revealed that voltage-dependent Ca²⁺ currents in AMC cells were suppressed by adenosine triphosphate, but not by muscarinic agonists. The fractionation analysis and immunocytochemistry indicated that Ca_V1.2 Ca²⁺ channels and M₄ receptors are located in the raft and non-raft membrane domains, respectively. We concluded that muscarinic stimulation in rat AMC cells does not produce voltage-dependent Ca²⁺ channel inhibition. This lack of muscarinic inhibition is at least partly due to physical separation of voltage-dependent Ca²⁺ channels and M₄ receptors in the plasma membrane.

I.  Introduction

Adrenal medullary chromaffin (AMC) cells as well as sympathetic ganglion neurons originate from the neural crest [17]. Thus, these two cells have many common excitability and signaling properties. Both of them receive innervation from sympathetic preganglionic nerve fibers. Acetylcholine (ACh) secreted from nerve terminals excites both cells via nicotinic and/or muscarinic ACh receptors (AChRs). In sympathetic ganglion cells, ACh produces fast and slow excitatory postsynaptic potentials (EPSPs) via nicotinic and muscarinic AChRs, respectively [11]. This dual mode of neuronal transmission has been preserved from frog to mammalian ganglion neurons [1, 9]. On the other hand, the mode of ACh-mediated neuronal transmission in AMC cells varies depending on the animal species [3, 37]: Nicotinic AChRs are mainly involved in ACh-mediated neuronal transmission in frog [36] and rat AMC cells [2, 25, 44], whereas muscarinic AChR involvement is assumed in chicken AMC cells [30].

One of the membrane properties characterizing rat ganglion neurons is muscarinic M₄ receptor-mediated inhibition of voltage-dependent Ca²⁺ channels [21]. Thus, ACh exerts a dual action of excitation and suppression in ganglion neurons: Excitation is due to M₁ receptor-mediated inhibition of M-type K⁺ channels [8, 31], and suppression is due to M₄ receptor-mediated inhibition of voltage-dependent Ca²⁺ channels [8, 21]. Because these two actions by muscarinic agonists exhibit a different concentration dependence [8], excessive ACh-mediated neuronal transmission is assumed to be prevented by M₄ receptor-mediated Ca²⁺ channel inhibition [31]. On the other hand, whether this M₄ receptor-mediated inhibition of voltage-dependent Ca²⁺ channels occurs in rat AMC cells remains unclear. In contrast to ganglion neurons, AMC cells lack neurites and secrete catecholamines and other chemicals in response to stimuli. Therefore, chemicals may function as auto/paracrine factors in AMC cells. Of note, adenosine triphosphate (ATP) is stored in chromaffin granules [46] and secreted together with catecholamines in response to stimuli [42]. Secreted ATP suppresses voltage-dependent Ca²⁺ channels via P2Y₁₂ receptors [15, 18, 32]. Because ATP-mediated inhibition functions as an efficient feedback mechanism to suppress excess excitation in AMC cells [19, 26], muscarinic signaling for Ca²⁺ channel inhibition, if present in AMC cells, might be redundant. The aim of the present study was to explore whether muscarinic inhibition of voltage-dependent Ca²⁺ channels occurs in rat AMC cells, and if it does not, to elucidate why this inhibition does not occur.

II.  Materials and Methods

Wister rats weighing 200–400 g (n = 25), which were obtained from Kudo (Tosu, Japan), were housed in cages at a controlled temperature (25°C) and exposed to a 12-hr photoperiod. The animals were given access to standard rodent chow and water ad libitum. All procedures for the care and treatment of animals were carried out in accordance with the Japanese Act on the Welfare and Management of Animals and the Guidelines for the Proper Conduct of Animal Experiments issued by the Science Council of Japan. The experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Occupational and Environmental Health (AE07-012) and complied with the principle outlined in the U.K. legislation on animal studies. All efforts were made to minimize suffering and to reduce the number of animals used in this study.

Whole-cell recording

The animals were killed by cervical dislocation and the adrenal glands were excised and immediately put into an ice-cold Ca²⁺-deficient balanced salt solution in which 1.8 mM CaCl₂ was simply omitted from standard saline. The standard saline contained 137 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.5 mM MgCl₂, 0.53 mM NaHPO₄, 5 mM D-glucose, 5 mM HEPES, and 4 mM NaOH (pH 7.4). The adrenal cortex was removed from the adrenal gland using microscissors and forceps under stereoscopic observation. The adrenal medulla was cut into two or three pieces and then incubated in a 0.5% collagenase-containing Ca²⁺-free solution at 36°C for 15 min. This enzymatic digestion was repeated twice while the preparations were gently stirred with 100% O₂ gas. After the incubation, the tissues were washed several times and kept in the Ca²⁺-free solution at 5°C until cell dissociation for current recordings. One or two pieces of the tissues were put into a bath apparatus, which was placed on an inverted microscope, and the AMC cells were dissociated mechanically with fine needles. Thereafter, the dissociated cells were allowed to adhere to the bottom for a few tens of minutes before the bath apparatus was perfused with saline at a rate of 1 mL min⁻¹. The whole-cell current was recorded in an isolated rat AMC cell by using the nystatin perforated patch method, as described elsewhere [27]. The current was recorded with an Axopatch 200A amplifier (Axon, Foster City, CA, USA) and then fed into a thermal recorder after low-pass filtering at 15 Hz and into a DAT recorder. To study voltage-dependent Ca²⁺ currents, pipettes were filled with a solution that contained 150 mM CsCl and 10 mM HEPES and was adjusted to pH 7.2 with CsOH. On the day of the experiment, nystatin dissolved in dimethyl sulfoxide (5 mg in 100 μL) was added to the pipette solution at a final concentration of 100 μg mL⁻¹ while the solution was being vortexed. All of the chemicals were bath applied. The experiments were carried out at 26 ± 2°C.

Fractionation analysis

Rat adrenal medullae were homogenized in a TNE/Tx buffer containing 0.5% Triton X-100, 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EGTA and a protease inhibitor cocktail (1 mM 4-(2-aminoethyl) benzene sulphonyl fluoride, 0.3 μM aprotinin, 2 μM E-64, 1 mM EDTA, and 2 μM leupeptin) (Calbiochem, San Diego, CA. USA) on ice. The homogenates were centrifuged at 500 g for 10 min at 4°C to remove the cell debris. The supernatant was subjected to fractionation with centrifugation in a discontinuous sucrose density gradient, as described previously [48]. The sucrose concentration of the homogenate was adjusted to 40% by adding 80% sucrose in TNE/Tx buffer. Then, TNE/Tx buffer with a discontinuous density gradient, consisting of 5%–30% sucrose in five steps, was layered over the lysate with 40% sucrose. The sample was centrifuged at 100,000 g for 18 hr. Seven fractions were collected from the top to the bottom of the gradient. The proteins were separated by 10% (w/v) sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane. To block nonspecific protein binding, the membrane was incubated in 5% (w/v) nonfat powdered milk dissolved in Tris-buffered saline with Tween 20 (TBS-T), which contained 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween 20. Then, the membrane was subjected to immunoblot analysis. First, it was incubated with one of the following primary antibodies (Abs): rabbit anti-caveolin-1 (sc-894: Santa Cruz Biotechnology, Santa Cruz, CA, USA) (RRID:AB_2072042), mouse anti-transferrin receptor (A11130: Molecular Probes, Eugene, OR, USA) (RRID:AB_2534136), mouse anti-M₄ (MAB1576: Chemicon, Temecula, CA, USA) (RRID:AB_2080217), rabbit anti-Ca_V1.2 (ACC-001: Alomone, Jerusalem, Israel) (RRID:AB_2039764), or rabbit anti-TWIK-related acid-sensitive K⁺1 (TASK1) (APC-024: Alomone) (RRID:AB_2040132). After washing in TBST, the membrane was incubated with the appropriate secondary Ab linked to horseradish peroxidase (Amersham, Buckinghamshire, UK). Finally, the protein bands were visualized by incubating the membrane with ECL-Plus (Amersham). Immunoblotting was repeated at least three times for each Ab.

Immunocytochemistry

After collagenase treatment, one or two pieces of adrenal medulla tissue were placed into a dish with non-fluorescent glass (P35GC-0-14-C: MatTek, Ashland, MA, USA) and then dissociated using fine needles. The dissociated AMC cells were fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.2) for 1 hr and then pre-incubated in PBS with 5% fetal bovine serum (FBS) (Nichirei, Tokyo, Japan) and 0.3% Triton X-100 for 30 min. For indirect immunofluorescence, the cells were incubated with mouse anti-caveolin-1 Ab (sc-53564: Santa Cruz) (RRID:AB_628859), rabbit anti-Ca_V1.2 Ab, and/or mouse anti-M₄ Ab. After incubation, the cells were washed three times in PBS and then incubated with the appropriate secondary Ab conjugated to Alexa Fluor 488 or 546 (Molecular Probes). The fluorescence was observed using a confocal laser scanning microscopy (LSM5 Pascal, Carl Zeiss, Tokyo, Japan). An oil-immersion objective lens with a magnification of 63× and a numerical aperture of 1.4 was used. For Alexa Fluor 488, a 488 nm laser was used and emission was observed at 510–560 nm (FITC-like fluorescence), whereas for Alexa Fluor 546, a 543 nm laser was used and emission was observed above 560 nm (rhodamine-like fluorescence). Immunocytochemical examination was repeated at least three times to confirm the reproducibility of the immunoreaction.

Cell culture and transfection

Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Tokyo, Japan) supplemented with 10% FBS. The LipofectAMINE 2000 reagent (Invitrogen) was used to transfect HEK293T cells with a construct encoding Ca_V1.2–super-enhanced yellow fluorescence protein (YFP), according to the manufacturer’s instructions. The transfected cells were placed onto glass coverslips coated with collagen type I (BD Biosciences, San Jose, CA, USA) and then cultured for 24 hr. The cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. After washing three times with PBS, the cells were incubated in PBS containing 0.1% Triton X-100 for 30 min and then with PBS containing 1% FBS for 1 hr at room temperature. The transfected cells were treated overnight with a rabbit anti-Ca_V1.2 Ab and then with goat anti-rabbit IgG Ab conjugated to Alexa Fluor 635. The coverslips were mounted in 50% glycerol containing 1 mg mL⁻¹ 4-diaminobenzene and immunostaining was observed with an LSM5 Pascal confocal laser scanning microscope. Ca_V1.2-like immunofluorescence and super-enhanced YFP were visualized with excitation at 633 nm and emission above 650 nm and with excitation at 514 nm and emission of 530 to 600 nm, respectively.

Perfusion experiment

The adrenal glands were removed from the rats under pentobarbital (60 mg kg⁻¹ intraperitoneal) anesthesia and then perfused retrogradely via the adrenal vein with saline at a rate of 0.15 mL min⁻¹ [45]. The glands were subjected to 40 min of recurrent perfusion with 1 mL of saline containing 10 μM Fluo-4 AM (Molecular Probes) and 0.2% Pluronic F 127 (Sigma-Aldrich, St. Louis, MO, USA), and then part of the adrenal cortex covering the medulla was carefully removed using microcissors. The adrenal gland was placed between one pair of silver circles for electrical stimulation, and then the gland was transferred to a chamber with the naked medulla on the glass bottom. The chamber was mounted onto the stage of the confocal laser scanning microscope and the adrenal gland was continuously perfused at 25–28°C. The sample was illuminated at 488 nm with an argon laser, and the emission above 510 nm was monitored. Fluorescence images were acquired every 5 sec. The extent of photobleaching was estimated by a curve that fit the intensities of responsive areas (up to eight areas in a frame) in the resting state with a polynomial function (at² + bt + c, where a, b, and c are constants and t is time). The intensity in each area was then corrected for photobleaching. A change in fluorescence intensity in response to electrical stimulation or an agonist was expressed as a fraction of the resting level. This relative value of change was averaged for each frame. The number of data represents the number of adrenal glands used in the perfusion experiments.

Statistics

All statistical analyses were performed with Sigma Plot (v. 13.0; Systat software, San Jose, CA, USA). The data are expressed as the mean ± standard error of the mean (SEM). When the data had a normal distribution (checked with the Shapiro–Wilk test), they were evaluated with two-tailed Student’s t-test. Otherwise, a non-parametric method as indicated was used. A P value of < 0.05 was considered statistically significant.

Source of agents

Muscarine chloride and oxotremorine sesquifumarate were obtained from Sigma-Aldrich; collagenase was from Yakult (Tokyo, Japan); and nicotine was from Nacalai (Kyoto, Japan).

III.  Results

Perfusion experiment

We examined whether muscarinic receptor activation regulates the excitability of AMC cells from the rat adrenal medulla, which was retrogradely perfused through the adrenal vein and loaded with the Ca²⁺ indicator Fluo-4. The transmural stimulation of the adrenal gland with 1.5-msec pulses of 50 V for 30 sec resulted in trans-synaptic activation of the AMC cells with a frequency-dependent increase in the Ca²⁺ signal (Fig. 1A and B). The relative amplitudes of such increases in response to electrical stimulation, which are expressed as a fraction of the resting level, are summarized in Fig. 1C. Application of 30 μM muscarine increased the Ca²⁺ signal, which comprised phasic and plateau components. This Ca²⁺ signal was caused by voltage-dependent Ca²⁺ influx, which was mediated by depolarization due to TASK channel inhibition [28, 35]. During the plateau phase of the Ca²⁺ signal, the increase in Ca²⁺ evoked electrically at 1 and 5 Hz did not change significantly (P > 0.05). On the other hand, when 30 μM GABA was applied, rat AMC cells exhibited an increase in Ca²⁺ signal with GABA_A receptor activation. As noted previously, there was a significant 10% decrease in electrically evoked Ca²⁺ during the GABA-induced increase in the Ca²⁺ signal due to a shunt effect of increased Cl⁻ conductance [29, 34].

Fig. 1.

Effects of muscarine and GABA on trans-synaptically evoked increase in the intracellular Ca²⁺ concentration in rat adrenal medullary chromaffin (AMC) cells. (A and B) Relative changes in Ca²⁺ signal in Fluo-4-loaded AMC cells are plotted against time. The adrenal gland was retrogradely perfused through the adrenal vein with saline. Nerve fibers remaining in the gland were electrically stimulated with 50 V pulses of 1.5 msec in duration at 0.5, 1, and 5 Hz during the indicated periods (bars). Muscarine (MUS) and GABA, each at 30 μM, were added to the perfusion solution during the indicated period (interrupted lines). Fluorescence intensities in up to eight areas in each frame were measured. After correction for the decline due to photobleaching, the increase in fluorescence intensity in response to electrical stimulation and muscarine or GABA is expressed as a fraction of the resting level (see the Materials and Methods). (C) Summary of the relative changes in Ca²⁺ signal in response to electrical stimulation at 0.5, 1, and 5 Hz. The data represent the mean ± standard error of the mean (SEM) of eight adrenal glands. (D and E) Relative amplitudes of the increase in the Ca²⁺ signal electrically evoked at 1 and 5 Hz during application of MUS and GABA, respectively. The relative amplitudes were obtained by dividing the relative changes in Ca²⁺ signal during MUS or GABA application with an average of these before and after its application. Statistical significance was evaluated by a paired Student’s t test. The trans-synaptically evoked increase in Ca²⁺ signal significantly decreased during application of GABA, but not MUS. The data represent the mean ± SEM of six and eight adrenal glands for MUS and GABA, respectively.

Effects on voltage-dependent Ca²⁺ channels

Voltage-dependent Ca²⁺ channel activity is inhibited by activation of purinergic, opioidergic, and β2 adrenergic receptors in rat AMC cells [14, 26] and by muscarinic M₄ receptor activation in rat sympathetic neurons [21]. The perfusion experiments, however, suggested that Ca²⁺ channel activity in rat AMC cells is not affected by muscarinic receptor stimulation. Therefore, we directly investigated whether muscarinic agonists inhibit voltage-dependent Ca²⁺ currents in dissociated rat AMC cells. As shown in Fig. 2, application of muscarine or oxotremorine at concentrations of up to 100 μM did not suppress voltage-dependent Ca²⁺ currents that were evoked by 70-mV pulses from the holding potential of −70 mV. On the other hand, application of up to 100 μM ATP resulted in the rapid suppression, as has been shown with L-type Ca²⁺ channel activity in rat AMC cells [26, 33]. Fig. 2C shows a summary of the relative amplitudes of voltage-dependent Ca²⁺ currents in response to 70-mV pulses in the presence of ATP, muscarine, or oxotremorine.

Fig. 2.

Lack of muscarinic receptor-mediated inhibition of voltage-dependent Ca²⁺ channels in rat AMC cells. (A and B) Muscarine (MUS) and adenosine triphosphate (ATP) did not and did produce a rapid inhibition of voltage-dependent Ca²⁺ currents in dissociated rat AMC cells, respectively. The amplitudes of voltage-dependent Ca²⁺ currents are plotted against the time. The whole cell current was recorded with the perforated path clamp method where the pipette was filled with CsCl (see the Materials and Methods). The holding potential was −70 mV, and 70 mV test pulses of 50 msec in duration were applied every 10 sec. The amplitudes of the current at the end of test pulses were measured with reference to the current level just before the test pulse. The insets (a, b, and c) show the current responses to the 70 mV test pulses. a, b, and c in the insets correspond to a, b, and c in the plots. (C) Summary of the amplitudes of voltage-dependent Ca²⁺ currents (I_Ca) in the presence of ATP, MUS, or oxotremorine (OXO) at concentrations of 30 to 100 μM. The I_Ca amplitudes during application of chemicals are expressed relative to the averaged values of I_Ca before and after it. The data represent the mean ± SEM (ATP, n = 7; MUS, n = 6: OXO, n = 4). Statistical significance was evaluated with the Mann–Whitney rank sum test.

Biochemical and immunocytochemical analysis

The failure of muscarine to inhibit voltage-dependent Ca²⁺ channels, half of which are the L-type in rat AMC cells [24, 26], suggests that muscarinic receptors are not closely associated with L-type Ca²⁺ channels in the plasma membrane in AMC cells. Because the Ca_V1.2 subtype of L-type Ca²⁺ channels is the predominant isoform in rat AMC cells [6], we explored the distribution of channels including Ca_V1.2 and receptors in the cell membrane both biochemically and immunocytochemically. First, we examined the immunospecificity of the anti-Ca_V1.2 Ab by using immunocytochemistry. As shown in Fig. 3A, YFP fluorescence of Ca_V1.2 fusion proteins exogenously expressed in HEK293T cells coincided with Ca_V1.2-like immunoreactive (IR) material, indicating that the Ab was immunospecific for Ca_V1.2.

Fig. 3.

Different distribution of Ca_V1.2 channel and muscarinic M₄ receptor in the cell membrane in rat AMC cells. (A) Immunostaining of Ca_V1.2-YFP fusion protein expressed in HEK293T cells with a rabbit anti-Ca_V1.2 antibody (Ab). HEK293T transfected with a Ca_V1.2-YFP construct were labeled with the rabbit anti-Ca_V1.2 Ab. a and b represent confocal images of Ca_V1.2-like immunofluorescence and YFP fluorescence, respectively; c represents a differential interference contrast (DIC) image. The immunoreaction and YFP fluorescence were visualized with excitation at 514 nm and emission of 530–600 nm and with excitation at 633 and emission above 650 nm, respectively. (B) Fractionation analysis of rat adrenal medullae for integral membrane proteins. The cell membrane was divided into the raft and non-raft membrane domains by using discontinuous sucrose density gradient centrifugation (see the Materials and Methods). The same volume of each fraction with 5%–40% sucrose was immunoblotted for caveolin-1, transferrin receptor (R), muscarinic M₄ receptor, and TASK1 channel. Note that caveolin-1, a raft membrane marker, was enriched in the 20% fraction, whereas transferrin R, a non-raft membrane marker, was present in the 40% fraction. (C) Double staining for caveolin-1 and Ca_V1.2 and for M₄ receptor and Ca_V1.2 in rat AMC cells. The first column indicates confocal images of caveolin-1 and M₄ receptor-like immunofluorescence. The second column shows confocal images of Ca_V1.2-like immunofluorescence. The third column is a merge of immunofluorescence images. The fourth column shows DIC images. The calibration applies to all the images. Dissociated rat AMC cells were treated overnight with rabbit anti-Ca_V1.2 Ab (dilution, 1:50) and mouse anti-caveolin-1 Ab (1:20) or mouse anti-M₄ Ab (1:50). Ca_V1.2 and caveolin-1 or M₄ receptor-like immunoreactive material were visible as rhodamine and FITC-like fluorescence, respectively. (D) Summary of the coincidence rates of caveolin-1 (Cav1) and M₄ with Ca_V1.2. The data represent the mean ± SEM (Cav1/Ca_V1.2, n = 10; M₄/Ca_V1.2, n = 5). Statistical significance was evaluated with an unpaired Student’s t test.

We next subjected proteins in rat AMC cells to fractionation analysis. As evident in Fig. 3B, sucrose density gradient centrifugation resulted in a clear separation of raft and non-raft membranes. Caveolin-1, a maker protein for the raft membrane domain [23], was mainly enriched in the fractions with 15%–20% sucrose, whereas the transferrin receptor, a marker protein for the non-raft membrane [23], was predominantly detected in the fraction with 40% sucrose (supplementary Fig. 1). The muscarinic M₄ receptors and TASK1 channels were detected only in the 40% sucrose fraction, whereas Ca_V1.2 was not recognized in any fraction. This failure of the anti-Ca_V1.2 Ab in immunoblotting might have been due to low immunoreactivity of the Ab we used. Therefore, we examined the distribution with immunocytochemistry. Fig. 3C shows that the majority of Ca_V1.2-like IR material coincided with caveolin-1-like IR material at the cell periphery, whereas M₄-like IR material scarcely overlapped with Ca_V1.2-like IR material. These results indicate that Ca_V1.2 and M₄ receptors are located in the raft and non-raft membrane domains in rat AMC cells, respectively.

IV.  Discussion

Lack of muscarinic inhibition of Ca²⁺ channels

Non-L-type Ca²⁺ channels, such as P/Q- and N-type, are known to be suppressed in a membrane-delimited manner by the stimulation of receptors coupled with G_i/o-type G proteins, such as M₂ [43], M₄ [21], and P2Y₁₂ receptors [15, 18]. In cultured rat AMC cells, however, there is unambiguous evidence that L-type Ca²⁺ channels containing Ca_V1.2 subunits are inhibited by β2 adrenergic agonists and ATP. This G protein-coupled receptor (GPCR)-mediated inhibition also occurs in a membrane-delimited manner [12, 39] and is sensitive to pertussis toxin [26], indicating the involvement of G_i/o-type G proteins. We found that in dissociated rat AMC cells, ATP inhibited voltage-dependent Ca²⁺ currents, as has been reported in previous studies, but muscarine did not. This failure of muscarinic stimulation was unexpected because the muscarinic M₄ receptor is consistently located at the cell periphery in the cells examined [25] and is generally coupled with G_i/o proteins [13]. Rat sympathetic neurons have been found to express M₁, M₂, and M₄ receptors at the messenger RNA (mRNA) and proteins levels [10], and M₄, but not M₂, is thought to be involved in the inhibition of voltage-dependent Ca²⁺ channels on the basis of its pharmacological properties [21]. This difference between AMC cells and sympathetic neurons regarding the inhibition of voltage-dependent Ca²⁺ channels suggests that AMC cells lack the molecular machinery to transduce the M₄ signal to Ca²⁺ channels. This possibility is supported by our biochemical and immunocytochemical findings. The fractionation analyses of receptors and channels revealed that M₄ receptors and TASK1 channels were enriched in the non-raft membrane fraction. On the other hand, we detected caveolin-1 in the raft fractions, and Ca_V1.2-like immunofluorescence coincided with caveolin-1-like immunofluorescence in AMC cells. Intriguingly, M₄ receptor-like immunofluorescence showed little overlap with Ca_V1.2-like immunofluorescence. Thus, the fractionation analysis and immunocytochemistry support the notion that Ca_V1.2 and M₄ receptors are located in the raft and non-raft membrane domains, respectively (Fig. 4).

Fig. 4.

Diagram showing localization of caveolin-1, Ca_V1.2, muscarinic M₄ receptor subtype, and TASK1 in the raft and non-raft membrane domains.

Caveolae are thought to be a platform for signal transduction, where GPCRs, G proteins, scaffolding proteins, and channels form a complex [5, 38, 41]. Muscarinic M₂ receptors, adenylate cyclase, G_i proteins, and Ca_V1.2 channels are concentrated in the raft membrane in mouse cardiac myocytes [7]. The exogenous expression of type-2 Na⁺/H⁺ exchange regulatory factor (NHERF2), a scaffolding protein, has been reported to reduce strongly the inhibition of N-type Ca²⁺ currents in sympathetic neurons by activation of either metabotropic glutamate mGluR5 receptors or nucleotide P2Y₁ receptors, probably by facilitating their coupling to G_q-mediated Ca²⁺ signaling [22]. In addition, the expression of STM1 in HEK293T cells recruits transient receptor potential cation channel subfamily C member 1 (TRPC1) to the raft membrane domain, thereby allowing TRPC1 to function as store-operated channels [4]. L-type [47] and non-L-type Ca²⁺ channels, such as Ca_V2.1 [16], are present in the raft membrane domain in a variety of cells, and the membrane-delimited inhibition of voltage-dependent Ca²⁺ channels is assumed to be due to the physically closed association of channels, G_i/o proteins, and metabotropic receptors, such as M₄ receptors. Thus, the lack of muscarinic inhibition of voltage-dependent Ca²⁺ channels in rat AMC cells suggests that rat AMC cells have no machinery to locate M₄ receptors in the raft membrane. Additional studies are required to gain a better understanding of the lack of the muscarinic inhibition.

Functional implications

The lack of muscarinic receptor-mediated inhibition of Ca²⁺ channels in AMC cells is reasonable from the functional point of view. In contrast to sympathetic ganglion neurons, AMC cells secrete catecholamines into the blood stream in response to neuronal inputs, thus functioning as endocrine cells. In mammals, AMC cells accumulate in the core of the adrenal gland and are surrounded by adrenal cortical cells [40]. Therefore, these morphological and functional features allow AMC cells to communicate with each other via auto/paracrine factors. As described earlier, ATP is secreted with catecholamines in AMC cells and suppresses voltage-dependent Ca²⁺ channels, thus functioning as an auto/paracrine factor [15, 20]. This negative feedback mechanism is rational for endocrine cells. Because AMC cells function as endocrine cells, it is important how they are regulated as a mass rather than how they are regulated individually. On the other hand, sympathetic ganglion neurons convey neuronal information to peripheral tissues in a point-to-point manner. Thus, the excitability needs to be regulated for each neuron.

It is worth noting that nicotine-induced secretion in frog AMC cells is suppressed by muscarinic stimulation [36]. In contrast to mammalian AMC cells, nicotinic AChR-mediated neuronal transmission is subjected to regulation by inhibitory muscarinic AChRs in frog AMC cells. This finding suggests that the endocrine function in frog AMC cells might be regulated in positive and negative manners by ACh released from the sympathetic preganglionic nerve terminals, as in sympathetic ganglion cells. This mode of regulation might not fit with our notion that endocrine cells are regulated as a mass. However, frog AMC cells do not form a mass; they are scattered among the adrenal cortical cells. Thus, cell-to-cell communication by auto/paracrine factors might not occur. It would be interesting to explore whether ATP is stored in chromaffin granules in frog AMC cells and whether it functions as an auto/paracrine factor.

To conclude, the trans-synaptically induced increase in Ca²⁺ signal in rat AMC cells was decreased by activation of GABA_A receptors, but not by that of muscarinic receptors. In addition, voltage-dependent Ca²⁺ currents in rat AMC cells were suppressed by ATP, but not by muscarinic agonists. These results indicate that muscarinic receptors are not involved in the inhibition of voltage-dependent Ca²⁺ channels in rat AMC cells. The fractionation analysis and immunocytochemistry revealed that Ca_V1.2 Ca²⁺ channels and muscarinic M₄ receptors are located in the raft and non-raft membrane domains, respectively. The failure of muscarinic receptor stimulation to inhibit voltage-dependent Ca²⁺ channels in rat AMC cells may be due, at least in part, to physical separation of muscarinic receptors and voltage-dependent Ca²⁺ channels in the plasma membrane. Rat AMC cells are subjected to inhibitory regulation of excitability by auto/paracrine factors, such as ATP, in contrast to sympathetic ganglion cells, in which ACh inhibits voltage-dependent Ca²⁺ channels via muscarinic M₄ receptors.

V.  Conflicts of Interest

The authors declare that there are no conflicts of interest.

VI.  Acknowledgments

The research reported in this publication was supported in part by a JSPS grant (17K0855 to M.I.). We appreciate Dr. Akira Warashina (Niigata University, Japan) for his contribution to the perfusion experiments. The construct encoding Ca_V1.2-super enhanced YFP was kindly provided by Dr. Masayuki Mori (University of Occupational and Environmental Health, Japan).

M.I. and K.H. designed the study, performed the experiment, analyzed the data, and wrote the manuscript. Both authors approved the final manuscript.

VII. References

• 1   Adams,  P. R. and  Brown,  C. A. (1982) Synaptic inhibition of the M-current: slow excitatory post-synaptic potential mechanism in bullfrog sympathetic neurones. J. Physiol. 332; 263–272.
• 2   Akiyama,  T.,  Yamazaki,  T.,  Mori,  H. and  Sunagawa,  K. (2003) Inhibition of cholinesterase elicits muscarinic receptor-mediated synaptic transmission in the rat adrenal medulla. Auton. Neurosci. 107; 65–73.
• 3   Alamo,  L.,  Garcìa,  A. G. and  Borges,  R. (1991) Electrically-evoked catecholamine release from cat adrenals. Role of cholinergic receptors. Biochem. Pharmacol. 42; 973–978.
• 4   Alicia,  S.,  Angélica,  Z.,  Carlos,  S.,  Alfonso,  S. and  Vaca,  L. (2008) STIM1 converts TRPC1 from a receptor-operated to a store-operated channel: moving TRPC1 in and out of lipid rafts. Cell Calcium 44; 479–491.
• 5   Allen,  J. A.,  Halverson-Tamboli,  R. A. and  Rasenick,  M. M. (2007) Lipid raft microdomains and neurotransmitter signaling. Nat. Rev. Neurosci. 8; 128–140.
• 6   Baldelli,  P.,  Hernández-guijo,  J. M.,  Carabelli,  V.,  Novara,  M.,  Cesetti,  T.,  Andrés-Mateos,  E., et al. (2004) Direct and remote modulation of L-channels in chromaffin cells: distinct actions on alpha1C and alpha1D subunits? Mol. Neurobiol. 29; 73–96.
• 7   Balijepalli,  R. C.,  Foell,  J. D.,  Hall,  D. D.,  Hell,  J. W. and  Kamp,  T. J. (2006) Localization of cardiac L-type Ca²⁺ channels to a caveolar macromolecular signaling complex is required for β₂-adrenergic regulation. Proc. Natl. Acad. Sci. U S A 103; 7500–7505.
• 8   Bernheim,  L.,  Mathie,  A. and  Hille,  B. (1992) Characterization of muscarinic receptor subtypes inhibiting Ca²⁺ current and M current in rat sympathetic neurons. Proc. Natl. Acad. Sci. U S A 89; 9544–9548.
• 9   Brown,  D. A. and  Selyanko,  A. A. (1985) Membrane currents underlying the cholinergic slow excitatory post-synaptic potential in the rat sympathetic ganglion. J. Physiol. 365; 365–387.
• 10  Brown, D. A., Buckley, N. J., Caulfield, M. P., Duffy, S. M., Jones, S., Lamas, J. A., et al. (1995) Coupling of muscarinic acetylcholine receptors to neuronal ion channels: closure of K⁺ channels. In “Molecular Mechanisms of Muscarinic Acetylcholine Receptor Function”, ed. by J. Wess, R. G. Landes Company, Austin, TX, USA, pp. 165–182.
• 11   Brown,  D. A. (2010) Muscarinic acetylcholine receptors (mAChRs) in the nervous system: some functions and mechanisms. J. Mol. Neurosci. 41; 340–346.
• 12   Carbone,  E.,  Carabelli,  V.,  Cessetti,  T.,  Baldelli,  P.,  Hernández-Guijo,  J. M. and  Giusta,  L. (2001) G-protein- and cAMP-dependent L-channel gating modulation: a manyfold system to control calcium entry in neurosecretory cells. Pflügers Arch. 442; 801–813.
• 13   Caulfield,  M. P. and  Birdsall,  N. J. (1998) International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol. Rev. 50; 279–290.
• 14   Cesetti,  T.,  Hernández-Guijo,  J. M.,  Baldelli,  P.,  Carabelli,  V. and  Carbone,  E. (2003) Opposite action of β1- and β2-adrenergic receptors on Ca_V1 L-channel current in rat adrenal chromaffin cells. J. Neurosci. 23; 73–83.
• 15   Currie,  K. M. and  Fox,  A. P. (1996) ATP serves as a negative feedback inhibitor of voltage-gated Ca²⁺ channel currents in cultured bovine adrenal chromaffin cells. Neuron 16; 1027–1036.
• 16   Davies,  A.,  Douglas,  L.,  Hendrich,  J.,  Wratten,  J.,  Van Minh,  A. T.,  Foucault,  I., et al. (2006) The calcium channel α₂δ-2 subunit partitions with Ca_V2.1 into lipid rafts in cerebellum: implications for localization and function. J. Neurosci. 26; 8748–8757.
• 17   Donoghue,  P. C.,  Graham,  A. and  Kelsh,  R. N. (2008) The origin and evolution of the neural crest. Bioessays 30; 530–541.
• 18   Ennion,  S. J.,  Powell,  A. D. and  Seward,  E. P. (2004) Identification of the P2Y₁₂ receptor in nucleotide inhibition of exocytosis from bovine chromaffin cells. Mol. Pharmacol. 66; 601–611.
• 19   Fernández,  A.,  Segura-Chama,  P.,  Albiñana,  E.,  Hernández-Cruz,  A. and  Hernández-Guijo,  J. M. (2010) Down-modulation of Ca²⁺ channels by endogenously released ATP and opioids: from the isolated chromaffin cell to the slice of adrenal medullae. Cell Mol. Neurobiol. 30; 1209–1216.
• 20   Fernández,  A.,  Segura-Chama,  P.,  Jiménez,  N.,  Carcía,  A. G.,  Hernández-Guijo,  J. M. and  Hernández-Cruz,  A. (2011) Modulation by endogenously released ATP and opioids of chromaffin cell calcium channels in mouse adrenal slices. Am. J. Physiol. Cell Physiol. 300; C610–C623.
• 21   Fernandez-Fernandez,  J. M.,  Wanaverbecq,  N.,  Halley,  P.,  Caulfield,  M. P. and  Brown,  D. A. (1999) Selective activation of heterologously expressed G protein-gated K⁺ channels by M₂ muscarinic receptors in rat sympathetic neurones. J. Physiol. 515; 631–637.
• 22   Filippov,  A. K.,  Simon,  J.,  Barnard,  E. A. and  Brown,  D. A. (2010) The scaffold protein NHERF2 determines the coupling of P2Y1 nucleotide and mGluR5 glutamate receptor to different ion channels in neuorns. J. Neurosci. 30; 11068–11072.
• 23   Francesconi,  A.,  Kumari,  R. and  Zukin,  R. S. (2009) Regulation of group I metabotropic glutamate receptor trafficking and signaling by the caveolar/lipid raft pathway. J. Neurosci. 29; 3590–3602.
• 24   García,  A. G.,  García-De-Diego,  A. M.,  Gandía,  L.,  Borges,  R. and  García-Sancho,  J. (2006) Calcium signaling and exocytosis in adrenal chromaffin cells. Physiol. Rev. 86; 1093–1131.
• 25   Harada,  K.,  Matsuoka,  H.,  Sata,  T.,  Warashina,  A. and  Inoue,  M. (2011) Identification and role of muscarinic receptor subtypes expressed in rat adrenal medullary cells. J. Pharmacol. Sci. 117; 253–264.
• 26   Hernández-Guijo,  J. M.,  Carabelli,  V.,  Gandía,  L.,  García,  A. G. and  Carbone,  E. (1999) Voltage-independent autocrine modulatoin of L-type channels mediated by ATP, opioids and catecholamines in rat chromaffin cells. Eur. J. Neurosci. 11; 3574–3584.I
• 27   Inoue,  M. and  Imanaga,  I. (1995) Mechanism of activation of nonselective cation channels by putative M4 muscarinic receptor in guinea-pig chromaffin cells. Br. J. Pharmacol. 114; 419–427.
• 28   Inoue,  M.,  Harada,  K.,  Matsuoka,  H.,  Sata,  T. and  Warashina,  A. (2008) Inhibition of TASK1-like channels by muscarinic receptor stimulation in rat adrenal medullary cells. J. Neurochem. 106; 1804–1814.
• 29   Inoue,  M.,  Harada,  K.,  Matsuoka,  H. and  Warashina,  A. (2010) Paracrine role of GABA in adrenal chromaffin cells. Cell Mol. Neurobiol. 30; 1217–1224.
• 30   Knight,  D. E. and  Baker,  P. F. (1986) Observations on the muscarinic activation of catecholamine secretion in the chicken adrenal. Neuroscience 19; 357–366.
• 31   Lechner,  S. G.,  Mayer,  M. and  Boehm,  S. (2003) Activation of M₁ muscarinic receptors triggers transmitter release from rat sympathetic neurons through an inhibition of M-type K⁺ channels. J. Physiol. 553; 789–802.
• 32   Lechner,  S. G.,  Dorostkar,  M. M.,  Mayer,  M.,  Edelbauer,  H.,  Pankevych,  H. and  Boehm,  S. (2004) Autoinhibition of transmitter release from PC12 cells and sympathetic neurons through a P2Y₁₂ receptor-mediated inhibition of voltage-gated Ca²⁺ channels. Eur. J. Neurosci. 20; 2917–2928.
• 33   Marcantoni,  A.,  Carabelli,  V.,  Comunanza,  V.,  Hoddah,  H. and  Carbone,  E. (2008) Calcium channels in chromaffin cells: focus on L and T types. Acta Physiol. (Oxf.) 192; 233–246.
• 34   Matsuoka,  H.,  Harada,  K.,  Endo,  Y.,  Warashina,  A.,  Doi,  Y.,  Nakamura,  J., et al. (2008) Molecular mechanisms supporting a paracrine role of GABA in rat adrenal medullary cells. J. Physiol. 586; 4825–4842.
• 35   Matsuoka,  H. and  Inoue,  M. (2017) Molecular mechanism for muscarinic M₁ receptor-mediated endocytosis of TWIK-related acid-sensitive K⁺ 1 channels in rat adrenal medullary cells. J. Physiol. 595; 6851–6867.
• 36  Nassar-Gentina, V., Luxoro, M. and Urbina, N. (1991) Cholinergic receptors and catecholamine secretion from adrenal chromaffin cells of the toad. Comp. Biochem. Physiol. 100C; 495–500.
• 37   Olivos,  L. and  Artalejo,  A. R. (2008) Muscarinic excitation-secretion coupling in chromaffin cells. Acta Physiol. (Oxf.) 192; 213–220.
• 38   Pani,  B. and  Singh,  B. B. (2009) Lipid rafts/caveolae as microdomains of calcium signaling. Cell Calcium 45; 625–633.
• 39   Pérez-Garci,  E.,  Larkum,  M. E. and  Nevian,  T. (2013) Inhibition of dendritic Ca²⁺ spikes by GABA_B receptors in cortical pyramidal neurons is mediated by a direct G_i/o-βγ-subunit interaction with Ca_V1 channels. J. Physiol. 591; 1599–1612.
• 40   Perry,  S. F. and  Capaldo,  A. (2011) The autonomic nervous system and chromaffin tissue: neuroendocrine regulation of catecholamine secretion in non-mammalian vertebrates. Auton. Neruosci. 165; 54–66.
• 41   Petralia,  R. S.,  Wang,  Y. X. and  Wenthold,  R. J. (2003) Internalization at glutamatergic synapses during development. Eur. J. Neurosci. 18; 3207–3217.
• 42   Rojas,  E.,  Pollard,  H. B. and  Heldman,  E. (1985) Real-time measurements of acetylcholine-induced release of ATP from bovine medullary chromaffin cells. FEBS Lett. 185; 323–327.
• 43   Shapiro,  M. S.,  Loose,  M. D.,  Hamilton,  S. E.,  Nathanson,  N. M.,  Gomeza,  J.,  Wess,  J., et al. (1999) Assignment of muscarinic receptor subtypes mediating G-protein modulation of Ca²⁺ channels by using knockout mice. Proc. Natl. Acad. Sci. U S A 96; 10899–10904.
• 44   Wakade,  A. R. and  Wakade,  T. D. (1983) Contribution of nicotinic and muscarinic receptors in the secretion of catecholamines evoked by endogenous and exogenous acetylcholine. Neuroscience 10; 973–978.
• 45   Warashina,  A. and  Inoue,  M. (2012) Ca²⁺ imaging in perfused adrenal medullae. J. UOEH 34; 163–173.
• 46   Winkler,  H. and  Westhead,  E. (1980) The molecular organization of adrenal chromaffin granules. Neuroscience 5; 1803–1823.
• 47   Xia,  F.,  Leung,  Y. M.,  Gaisano,  G.,  Gao,  X.,  Chen,  Y.,  Fox,  J. E. M., et al. (2007) Targeting of voltage-gated K⁺ and Ca²⁺ channels and soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins to cholesterol-rich lipid rafts in pancreatic α-cells: effects on glucagon stimulus-secretion coupling. Endocrinology 148; 2157–2167.
• 48   Yuyama,  K.,  Sekino-Suzuki,  N.,  Sanai,  Y. and  Kasahara,  K. (2007) Translocation of Activated Heterotrimeric G Protein Gα_o to Ganglioside-enriched Detergent-resistant Membrane Rafts in Developing Cerebellum. J. Biol. Chem. 282; 26392–26400.